Abstract
Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.
Highlights
Intracellular bacteria invade eukaryotic cells, followed by subversion of endocytic pathway, which results in formation of membrane-bound phagosomes
Proteins were transferred to nitrocellulose membrane in Transfer Buffer (Tris Base, Glycine, Methanol, dd H2 O) and blocked for 1 h at room temperature in 1x Tris Buffered Saline (TBS, SigmaAldrich, USA) with 0.1% (w/v) Tween-20 (TBST, Sigma-Aldrich, USA) and 3% (m/v) Bovine Serum Albumine (BSA, SigmaAldrich, USA)
It is essential for Francisella to replicate within host cells to successfully establish an infection and cause disease
Summary
Intracellular bacteria invade eukaryotic cells, followed by subversion of endocytic pathway, which results in formation of membrane-bound phagosomes. They are cable to modulate the membrane protein and lipid composition of phagosomes. The study of Francisella vacuolar escape kinetics has brought controversy to the field varying from the 15 min to 8 h post-infection (Golovliov et al, 2003; Clemens et al, 2004; Santic et al, 2005, 2008; Checroun et al, 2006; McCaffrey and Allen, 2006). These differences are likely due to variation in the host cells used, the Francisella species and the methodological approaches used by various studies
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